Heat‐Activatable Primers for Hot‐Start PCR: Oligonucleotide Synthesis and Basic PCR Setup

Abstract

2'-Deoxyribonucleoside-3'-O-(4-oxotetradec-1-yl) phosphoramidites (OXT phosphoramidites) are used to prepare modified oligodeoxyribonucleotide primers containing heat cleavable OXT phosphotriester protecting groups at 3'-ultimate and penultimate internucleotide linkages. The OXT-modified primers significantly improve performance of the polymerase chain reaction (PCR) compared to standard DNA primers by substantially reducing or eliminating the accumulation of PCR artifacts such as dimerized primers and misprimed amplicons. Basic protocols for synthesis of OXT-modified oligonucleotide primers and for performing hot-start PCR are described.